The oil palm belongs to the genus Elaeis which contains two species, E. guineensis and E. oleifera. It is regarded as the most efficient oil bearing crop in the world out yielding all other crops of the same genre, e.g., soybean, rapeseed and sunflower. The ability to produce oil at an average yield of 3.74 tonne/ha/year, on land 10 times smaller than the requirement for soybean (Oil World, 2007) and with a productive life cycle of 25-30 years, makes the oil palm a lucrative agricultural crop. However, of late the oil yield has reached stagnation. Nevertheless, demand for edible oils is predicted to escalate to feed the growing world population.
The oil palm has gone through at least two known cycles of yield improvements since its introduction as an oil crop in Malaysia, the first wave being the introduction of the hybrid tenera (DxP), which replaced the dura as commercial planting material. This demonstrated an increase in oil yield of up to 30% by merely manipulating a single gene (Kushairi et al., 2006; Singh et al., 2013). However, the average oil yield in Malaysia has hovered between 3.5 and 3.9 t/ha/yr for the last two decades. Having dropped to the number two spot in palm oil production, Malaysia—and all other palm oil producing countries—is in need of yield improvement. This is further compounded by the fact that agricultural land is becoming a rarity. Therefore increased production by planting larger areas is no longer seen as an alternative.
Through years of breeding and selection, the palm oil industry has already produced palms yielding as high as 13.6 t/ha/yr (Sharma and Tan, 1999) which are close to the theoretical yield of 18.2 t/ha/yr (Corley, 1998). The best experimental plot has produced an average of 9.8 t/ha/yr of palm oil (Musa and Gurmit, 2008) with selected progenies able to achieve up to 12.2 t/ha/yr (Raj anaidu et al., 1990). Cloning these super palms may provide the industry with the much-needed high-yielding planting materials to get it out of the stagnation rut. Hence, clones for commercial use are touted as the second wave of crop improvement for the oil palm.
Due to its biological structure, the oil palm has no natural means of vegetative propagation and conventional hybrid breeding methodology would require at least three generations, or over 20 years, to realize such superior yields (Soh et al., 2005). Successful vegetative propagation of oil palm was first described in the 1970s (Jones, 1974; Rabechault and Martin, 1976). Jones (1995) gave a rather comprehensive and personal account of its development. These successful reports of oil palm cloning prompted the development of tissue culture laboratories to provide clonal oil palm planting material. Encouraging results from early field trials set the pace for more laboratories to follow suit. By the mid-1980's, there were already 10 clonal oil palm laboratories in Malaysia (Wooi, 1990) and others elsewhere (Le Guen et al., 1991).
However, when Corley et al. (1986) reported the mantling phenomenon for the first time, the whole clonal industry led by the pioneering Bakasawit/Unifield and Tropiclone commercial laboratories decided to cut back on production and reverted to research and development. The then, Palm Oil Research Institute of Malaysia (PORIM), now known as Malaysian Palm Oil Board (MPOB), as the custodian of the palm oil industry, was assigned the task of spearheading research in clonal abnormalities.
Through a concerted effort, by the early 1990's, the results obtained suggested that better tissue culture protocols needed to be established, which included subculturing procedures and the use of less devastating types of growth regulators. Alternative methods were also proposed such as suspension and protoplast cultures as a means to avoid subculturing. Cloning of dura and pisifera parents, followed by conventional crossing to circumvent the potential occurrence of somaclonal variants from clonal teneras, was amongst the different methods discussed (Ong-Abdullah, Viva 562/2011). Interestingly, up to 10% of abnormal palms spontaneously reverted to normal and remained normal for some time (Durand-Gasselin et al., 1990). Seedlings developed from Mantled fruits e.g., clone 115E, were normal; refuting the possibility that abnormality is due to a dominant gene effect or to maternally transmitted factors. Through conventional genetic crossings conducted by Rao and Donough (1990), this trait was also shown to behave in a non-Mendelian manner.
Earlier attempts that employed techniques such as flow cytometry, random amplified polymorphic DNA (RAPD) or the classical amplified fragment length polymorphisms (AFLP) analysis failed to yield any detectable differences between Mantled and normal palms (Rival et al. 1997, 1998; Matthes et al. 2001). However, when methylation sensitive or related technologies were utilized, the methylation level of the Mantled genome appeared to be altered (Jaligot et al. 2002, Matthes et al. 2001, Jaligot et al. 2004).
Subsequently, further research concentrated on understanding the underlying molecular cause(s) and epigenetic regulation of mantling. It was also known that in Mantled oil palms, staminodes and stamens of pistillate and functional flowers develop respectively as pseudocarpels (Morcillo et al., 2006). In severe cases, the flowers are sterile with abortive fruits leading to lower yields. It was postulated that since homeotic modifications had taken place, it was highly likely that the B-function homeotic MADS box genes of the ABCDE model for flower organ identity (Murai, 2013) are involved.
Following the MADS box hypothesis, MADS-box containing genes from the oil palm were isolated (Alwee et al., 2006; Auyong, 2006) using the MADS box-directed profiling technique (van der Linden et al. 2002). This method allows the visualization of DNA polymorphisms in restriction sites at the MADS box vicinity among normal, abnormal and reverted oil palms. Two markers, namely MM77 and MM78 (EP Patent Appl. No. 13162130.2) were identified and the latter was widely used for further validation although it was found not to fall in the class of MADS box genes. In the course of validating MM78 and from past experiences with other unrelated markers, it was confirmed that the functional use of these markers is genotype dependent. Therefore, they have little or no use when tested on clones from other genetic backgrounds. This has been the main point of contention in biomarker development for clonal fidelity of the oil palm.
Previous studies have found an overall decrease in DNA methylation in mantled palms relative to ortets and normal ramets (Jaligot et al. 2000; Matthes et al. 2001; Jaligot et al. 2002; Jaligot et al. 2004). These results are similar to observations in Arabidopsis and other plant cell cultures, in which transposable elements (TEs) are hypomethylated and expressed (Tanurdzic et al. 2008; Miguel et al. 2011; Castilho et al. 2000; Kubis et al. 2003). In addition to TEs, somaclonal regenerants in rice and maize undergo extensive gene and promoter hypomethylation (Stroud et al. 2013; Stelpflug et al. 2014), which might also contribute to somaclonal variation in oil palm and other crops. The homeotic transformations observed in mantled palms resemble defects in B-function MADS box genes, suggesting that retroelements within one or more MADS box genes, or the MADS box genes themselves are candidates for epigenetic modification (Adam et al. 2005). However, decades of research into DNA methylation changes in candidate retroelements (Castilho et al. 2000; Kubis et al. 2003; Jaligot et al. 2014) and candidate homeotic genes (Syed Alwee et al. 2006; Adam et al. 2007; Jaligot et al. 2014) have yet to identify epigenetic changes that are consistently found in somaclonal mantled palms. And indeed, recent studies of rice and Arabidopsis plants regenerated from tissue culture implicate genetic rather than epigenetic mechanisms as being responsible for somaclonal variation (Jiang et al. 2011; Miyao et al. 2012.